The present invention relates in general to enzymatic diagnostic reagents and methods of use, and in particular to stabilizers for coenzymes, especially for NADH- or NADPH-containing reagents for CO.sub.2 detection.
The reduced forms of various compounds, e.g., nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), are coenzymes, as the hydrogen donors, for reactions catalyzed by many enzymes, e.g., dehydrogenases. Similarly, the oxidized forms of coenzymes are hydrogen acceptors for other enzymatic reactions. These enzymatic reactions requiring coenzymes have a wide variety of uses in pharmacology, chemical syntheses, and clinical diagnostics.
In particular, reactions which either consume or produce NADH or NADPH are easily detectible by measuring the UV absorbance of solutions containing them at about 340 nm, the absorption maximum of NADH and NADPH. These reactions can be used to directly measure the presence of an analyte in the solution. For example, the presence of a substance which is either a substrate for, or an enzyme which is a catalyst for, a particular reaction can be detected by adding it to a diagnostic agent comprising either the enzyme for the reaction, or the substrate for the reaction, respectively, in the presence of a given amount of a coenzyme required for the reaction, and measuring the appearance or disappearance of the reduced or oxidized form of the coenzyme.
Alternatively, a first enzyme reaction which is to be detected, but which does not produce or consume detectable reagents, may be indirectly coupled to a second reaction which does produce detectable products, by having in the diagnostic reagent a second enzyme/coenzyme combination which can be used to detect the appearance or disappearance of a substrate for the second enzyme which is produced or consumed during the reaction of the first enzyme upon it. Thus, for example, in order to measure the presence of the very important enzyme SGOT (serum glutamic-oxaloacetic transaminase, which enzyme is released into the blood after a myocardial infarct), the diagnostic agent contains aspartate and .alpha.-ketoglutarate, which are converted by SGOT to glutamate and oxaloacetate. ##STR1## However, none of these compounds is easily detectable spectrophometrically, or by other simple methods. Therefore, in order to detect this first reaction, it is coupled to another reaction in which the thus-formed oxaloacetate is converted to malate by a second enzyme, malate dehydrogenase, with the concomitant oxidation of NADH into NAD.sup.+, this second reaction being detectable spectrophotometrically by the disappearance of NADH: ##STR2## This indirect method of measurement thus allows detection of analytes which are not themselves either substrates or enzymes involved in NADH production or consumption.
However, a major and thus-far unsatisfactorily-resolved problem in this field is that the coenzymes for these reactions, in addition to being oxidized, e.g., to NAD.sup.+ and NADP.sup.+, respectively, during the reactions proper, are very susceptible to undesired oxidation, by, e.g., dissolved oxygen, as well as to decomposition of the oxidized form, prior to use, both when the compounds are in dry form and when they are in solution. In particular, NADH and NADPH are known to be unstable in solution, especially under acidic conditions (Wu et al., Clin. Chem. 32, 314-319 (1986); Lowry et al., J. Biol. Chem. 236, 2756-2759 (1961); Burton et al., Arch. Biochem. Biophys. 101, 150-159 (1963)).
The stability of these coenzymes in diagnostic reagents is of particular importance, as is evident by the many attempts in the prior art to stabilize them, again, both in dry powder form as well as in solution. These previous attempts have included stabilizing the coenzyme test composition using sulfhydryl-containing compounds (U.S. Pat. No. 3,746,625), other special stabilizers (Chem. Abstr. Vol. 104, No. 11, Sec. 109, Abstr. No. 084922), or preserving the coenzymes in an organic solvent matrix (U.S. Pat. No. 4,277,562). The most successful approach has been to maintain the coenzymes at an alkaline pH (e.g., at a pH &gt;8.0) with a suitable buffer (Wu, supra). However, no effective method has been suggested to maintain a stable NADH or NADPH solution at a pH below 8.0, especially in buffers that have a high concentration of phosphate anion (Wu, supra).
It is also acknowledged that the reductive regeneration of NADH and NADPH via enzymatic, chemical or other physical methods is well known in organic syntheses and bioreactors in order to decrease the cost incurred by continually adding large amounts of the expensive reduced coenzymes (Chenault et al., Appl. Biochem. Biotech. 4, 147-197 (1987); Wong et al., J. Am. Chem. Soc. 103, 4890-4899 (1981); Wong et al., J. Am. Chem. Soc. 107, 4028-4031 (1985); Suye et al., Enz. Microb. Technol. 7, 418-424 (1985); U.S. Pat. No. 4,766,071; Wang et al., Biochem. Eng. 12 119-146 (1979)). However, such regeneration would have been expected to be detrimental to the stabilizing of diagnostic reagents which utilize coenzymes which depend upon the quantitative determination of NADH as a means of detection. This is because when the production or disappearance of the coenzyme is used to measure the concentration of the diagnostically significant analyte, the addition of NAD.sup.+ -reducing enzymes would be expected to seriously distort the results of those diagnostic tests, by changing the levels of the very reactant which is to be measured.